The ribosome, the machinery essential for protein synthesis, obviously plays a key role in cell growth and its synthesis is intimately connected to the regulation of cell proliferation both in prokaryotic and eukaryotic cells. When cells are stimulated to proliferate by various means, an increase in ribosome synthesis takes place as one of the very early events before actual cell proliferation occurs. Our goal is to understand how cells regulate the production of ribosomes in response to environmental conditions. It is known that synthesis of ribosomal RNA (rRNA) is central in determining overall synthesis of ribosomes, and hence, we will concentrate on regulation of rRNA synthesis. We use the yeast Saccharomyces cerevisiae as a model system because of the ease of genetic manipulation and the available knowledge of rRNA synthesis by RNA polymerase I. We will continue to study interactions of components of the RNA polymerase I transcription machinery with the rDNA template both in vitro and in vivo. To elucidate the mechanisms of regulation, we will analyze the state of these components and the rDNA template in cells with high and low rRNA synthesis rates. Chromatin immunoprecipitation assay, in vitro rDNA transcription assay of extracts and electron microscopic analysis of rDNA chromatin spreads will be used for this purpose. Genetic approaches will also be used to identify components involved in the regulation. The genes for rRNAs are tandemly repeated in the nucleolus, which occupies a specific region in the nucleus separate from other chromosomal genes. This is reflected in the phenomenon of silencing of reporter genes for RNA polymerase II, and we will study it in relation to the state of rDNA genes. The nucleolus is obviously important for cell growth as the site of ribosome synthesis, but its structure is complex and its functional importance may not be fully appreciated. In fact, recent studies suggest its importance in regulation of cell cycle and possibly in cell aging. We will use genetic approaches to examine the importance of the copy number of rDNA genes, DNA elements within rDNA and chromosomal position of rDNA genes in nucleolar structure and function. We expect that the proposed work will make significant contributions to our understanding of important nucleolar functions including ribosome synthesis and its regulation.